Abstract
Hydroxytyrosol derivatives are the most important phenolic components in virgin olive oil due to their well-demonstrated biological activities. In this regard, two phenyl acetaldehyde reductase genes, OePAR1.1 and OePAR1.2, involved in hydroxytyrosol synthesis, have been identified from an olive transcriptome. Both genes were synthesized and expressed in Escherichia coli, and their encoded proteins were purified. The recombinant enzymes display high substrate specificity for 2,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) to form hydroxytyrosol. The reaction catalyzed by OePAR constitutes the second, and last, biochemical step in the formation of hydroxytyrosol from the amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) in olive. OePAR1.1 and OePAR1.2 enzymes exhibit high thermal stability, similar pH optima (pH 6.5), and high affinity for 3,4-DHPAA (apparent Km 0.6 and 0.8 µmol min−1 mg−1, respectively). However, OePAR1.2 exhibited higher specific activity and higher expression levels in all the olive cultivars under study. The expression analyses indicate that both OePAR1.1 and OePAR1.2 genes are temporally regulated in a cultivar-dependent manner. The information provided here could be of interest for olive breeding programs searching for new olive genotypes with the capacity to produce oils with higher levels of hydroxytyrosol derivatives.
Highlights
The most relevant phenolic compounds in olive (Olea europaea L.) belong to the secoiridoids family [1]
Three of those olive transcripts putatively annotated as cinnamyl-CoA reductases (olive genome database https://denovo.cnag.cat/olive_data, OE6.OLIVEFAT accession numbers: OE6A039209, OE6A025790 and OE6A018558) showed homology to Rr4HPAR1 with high percentages of identity (88–90%), and high statistical significance (Table S1)
Three other transcripts (OE6.OLIVEFAT accession numbers: OE6A068874, OE6A030751 and OE6A074153P) showed high homology to Rr4HPAR2 and low e-values (Table S1), but were discarded as candidate genes to control the final step of tyrosol and hydroxytyrosol biosynthesis since, according to Torrens-Spence et al [25], the Rr4HPAR2 enzyme has very low activity toward hydroxyphenyl aldehydes as substrate, such as the 4-HPAA, compared to Rr4HPAR1
Summary
The most relevant phenolic compounds in olive (Olea europaea L.) belong to the secoiridoids family [1]. During the industrial process to obtain virgin olive oil (VOO), the secoiridoid glucosides, initially present in the olive fruit tissues, are hydrolyzed by β-glucosidases into secoiridoid derivatives less hydrophilic than the originals, and making them partially soluble in the oil matrix [2,3]. These secoiridoid derivatives are mainly the aldehyde and dialdehyde forms of oleuropein and ligstroside aglycones which are by far the major phenolic compounds in VOO [4]. The scientific evidences on the health benefits of hydroxytyrosol have led the European Union to approve a health claim on olive oil polyphenols which may be applied to those oils containing at least 250 ppm of hydroxytyrosol and its derivatives [11]
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